阅读说明：本技术 一种降低肌球蛋白-5蛋白及其在氰基丙烯酸酯类药物抗性治理中的应用 (Myosin-5 protein reduction and application thereof in cyanoacrylate drug resistance treatment ) 是由 周明国 宋修仕 谷凯鑫 侯毅平 段亚冰 王建新 于 2018-05-28 设计创作，主要内容包括：本发明提供肌球蛋白5基因Myo5区段在防治植物真菌病害和/或增强植物的抗病性中的应用。所述肌球蛋白5基因来源于禾谷镰刀菌、亚洲镰刀菌、水稻恶苗病菌、枯萎病菌、稻瘟病菌、灰霉病菌、棉花黄萎病菌或油菜菌核病菌。所述肌球蛋白5基因Myo5区段是以Myo5dsRNA区段、dsRNA区段的联合或以全长或部分Myo5dsRNA进行RNase消化后随机产生15-30nt的siRNA。本发明还提供含有前述肌球蛋白5基因Myo5区段的体外干扰制剂。本发明的肌球蛋白-5Myo5基因RNA干扰技术具有增加病原真菌的药敏性、降低抗药性水平、干扰致病力、增强植物抗病性和防治植物病害的特异性等绿色、安全的突出优点。(The invention provides application of a myosin5 gene Myo5 segment in preventing and treating plant fungal diseases and/or enhancing disease resistance of plants. The myosin5 gene is derived from fusarium graminearum, fusarium asiaticum, rice bakanae disease, fusarium wilt, rice blast, botrytis cinerea, verticillium dahliae or sclerotinia sclerotiorum. The myoglobulin 5 gene Myo5 segment is a 15-30nt siRNA generated randomly by RNase digestion with a Myo5dsRNA segment, a combination of dsRNA segments or full-length or partial Myo5 dsRNA. The invention also provides in vitro interfering agents containing the myoglobulin 5 gene Myo5 segment described above. The RNA interference technology of the myosin-5 Myo5 gene has the outstanding advantages of increasing the drug sensitivity of pathogenic fungi, reducing the drug resistance level, interfering the pathogenicity, enhancing the disease resistance of plants, preventing and treating the specificity of plant diseases and the like, and is green and safe.)

1. The myosin5 gene Myo5 segment can be used for preventing and controlling plant fungal diseases and/or enhancing the disease resistance of plants.

2. Application of myosin5 gene Myo5 segment in inhibiting pathogenic fungi development and pathogenicity.

3. Use of the Myo5 segment of the myosin5 gene to increase the drug sensitivity of pathogenic fungi to myosin inhibitors.

4. Use according to one of claims 1 to 3, characterized in that the myosin5 gene is derived from Fusarium graminearum (Fusarium graminearum), Fusarium asia (F.asiaticum), Fusarium graminearum (F.moniliforme), Fusarium oxysporum (F.oxysporum), Magnaporthe oryzae (Magnaporthe oryzae), Botrytis cinerea (Botrytis cinerea), Verticillium dahliae (Verticillium daiiae) or Sclerotium brasiliensis (Sclerotium sclerotiorum).

5. Use according to one of claims 1 to 3, characterized in that the myosin5 gene Myo5 segment is randomly generated with siRNA 15-30nt after RNase digestion with Myo5dsRNA segment, combination of dsRNA segments or with full or partial Myo5 dsRNA.

6. The use of claim 4, wherein the Myo5dsRNA segment is obtained by dividing cDNA of Myo5 gene into 8 different segments, each segment being named Myo5-1(cDNA start and stop site 1nt-473nt), Myo5-2(cDNA start and stop site 456nt-938nt), Myo5-3(cDNA start and stop site 939nt-1455nt), Myo5-4(cDNA start and stop site 1381nt-1915nt), Myo5-5(cDNA start and stop site 1828nt-2313nt), Myo5-6(cDNA start and stop site 2253nt-2739nt), Myo5-7(cDNA start and stop site 2649nt-3206nt) and Myo5-8, wherein, when Myo5 gene is derived from Fusarium graminearum or Fusarium asiae, the cDNA start and stop site corresponding to Myo5-8 segment is 3149-3645, and when Myo5 gene is derived from Fusarium graminearum or Fusarium asia The starting and stopping site of cDNA corresponding to the Myo5-8 segment is 31nt-final base when Fusarium oxysporum (F.oxysporum), Magnaporthe oryzae (Magnaporthe oryzae), Botrytis cinerea (Botrytis cinerea), Verticillium dahliae (Verticillium dahliae) or Sclerotium sclerotiorum (Sclerotinia sclerotiorum) is used.

7. The use according to claim 3, wherein the combination of dsRNA segments is a gene segment obtained by using an RNAi Kit, using a combination of a plurality of dsRNA segments according to claim 6 as a template for synthesis of the segment.

8. An in vitro interference preparation comprising the myosin5 gene Myo5 segment of claim 5.

9. The use of the in vitro interference preparation of claim 8 in the breeding of disease-resistant varieties of transgenic plants.

[ technical field ] A method for producing a semiconductor device

The invention belongs to the technical field of genetic engineering, and relates to fusarium myosin-5 gene Myo5, an RNAi vector of the gene, in-vitro preparation of the gene Myo5dsRNA and application of the gene in preventing and treating plant diseases.

[ background of the invention ]

The cyhalothrin is a novel cyanoacrylate myosin inhibitor independently developed in China, and has the advantages of strong bactericidal activity, high specialization, capability of reducing fusarium DON toxin pollution, yield increase effect on plants and the like. At present, the market share of the bactericide is continuously increased, and the bactericide becomes one of high-efficiency selective bactericides for preventing and treating important diseases of main crops such as wheat scab (Fusarium spp.), rice bakanae disease (f.fujikuroi), vegetable root rot (Fusarium spp.) and the like. Although a drug-resistant strain of plant pathogenic fusarium to the cyanamide is not found in the field at present, the target pathogenic fungi are easy to generate drug resistance through ultraviolet mutagenesis and medicament domestication under laboratory conditions, wherein the proportion of the anti-neutralization high-resistance strain is large, and the fitness is high, so that the myosin inhibitor has high resistance risk.

Research shows that the main reason for the drug resistance of wheat scab (Fusarium spp.) to the cyanamide is the change of the affinity of the bactericide and target protein caused by the mutation of the molecular target myosin of the bactericide. Studies have shown that mutations in the cyanamide target myosin-5 gene at least at codons encoding amino acids 135, 151, 204, 216, 217, 418, 420, 424, 434, 577, 580 and 581 can produce different levels of drug resistance.

In recent decades, RNA interference has become a hot spot of scientific research, the technology obtains a large amount of results in the aspect of disease control, and the disease can be effectively prevented by specifically interfering key genes of pathogenic bacteria in a host-induced gene silencing (HIGS) or in vitro and in vitro interference mode. The resistance of wheat and barley to powdery mildew can be improved by interfering with the Avra10 gene of Blumeria graminis (Blumeria) by HIGS technology (Nowara et al, HIGS: host-induced gene cloning in the objective biological genetic mapping. plant cell 2010 (22): 3130) -3141). Tinoco et al constructed stem-loop constructs of GUS and transformed tobacco, and found that transgenic tobacco could inhibit the expression of Fusarium moniliforme (Fusarium moniliforme) GUS gene (Tinoco et al, In vivo trans-specific gene cloning In fungal cells by In plant expression of a double-stranded RNA. BMC biology.2010 (8): 27). Hu et al expressed dsRNA of Fusarium oxysporum FOW2, FRP1 and OPR in Arabidopsis, so that Fusarium oxysporum invaded transgenic plants at a significantly lower rate than controls, and three genes FOW2, FRP1 and OPR in Fusarium oxysporum were interfered with to different degrees (Hu et al, Down-regulation of Fusarium oxysporum end genes by y host-delayed RNA interference resistance genes. frontiers in chemistry.2015.(3): 1-10).

The target gene for RNA interference is the key to determine the magnitude of the RNA interference effect. The myosin-5 gene (or written as myosin-5 gene, myosin-5, myosin5, myo-5, myo5) belongs to myosin family gene, and its coded protein is a motor protein which utilizes ATP hydrolysis to provide kinetic energy for cell, and is an important component of cytoskeleton, and can be used for participating in biological processes of cell substance exchange, signal transmission and cell polarity, etc. The myosin-5 gene exists in a single copy form in most plant pathogenic fungi and is an indispensable key functional gene for the fungi to maintain life activities, and the knockout of the gene can cause the fungal cell death phenomenon. Therefore, the reduction of the expression of the pathogenic fungus myosin-5 gene by a molecular biology technical means not only can increase the sensitivity of the plant pathogenic fungus to a myosin inhibitor and reduce the drug resistance level, but also can interfere the normal growth and development of the fungus and reduce the pathogenicity. Meanwhile, the myosin-5 gene has larger genetic differentiation in different species, RNAi is designed aiming at myosin-5 gene sequences of different fungal species, and corresponding fungal diseases can be specifically controlled. Thus, the myosin-5 gene is an excellent target for RNA interference.

[ summary of the invention ]

The invention aims to provide an effective RNA interference section of a pathogenic fungus myosin-5 gene (Myo5) for enhancing drug sensitivity of fungi to myosin inhibitors or reducing drug resistance and plant disease resistance aiming at the myosin-5 gene (Myo5), so that the effective RNA interference section can be applied to plant disease resistance and in-vitro interference; another objective is to develop a Myo5dsRNA nucleic acid pesticide for controlling plant diseases caused by fungi and increasing the drug sensitivity of the fungi to myosin inhibitors. The Myo5 gene and its segment can be used as the target of host induced gene silencing and in vitro interference, and is sprayed on the surface of host plant by using in vitro interference method, or is introduced into plants (including wheat, corn, rice, strawberry, grape, etc.) by gene engineering means such as transgene, plant virus, etc., and silences the myosin-5 gene expression of fungi by RNAi approach, thereby reducing the pathogenicity of fungi, enhancing the drug sensitivity to Cyanoxastrobin, reducing the drug resistance and enhancing the disease resistance of plants, and providing a novel subversive technology for specifically controlling plant diseases.

In order to achieve the purpose, the invention aims at the myosin-5 gene sequence to construct RNAi carrier, and screens effective RNAi section to reduce pathogenic bacteria pathogenicity, thereby reducing disease occurrence; according to the principle of gene interference, the RNA interference mechanism that the Myo5dsRNA is sprayed or the Myo5RNAi plant is constructed by utilizing a transgenic technology, and the myosin-5 gene can be directly or (and) indirectly induced by fusarium, rice blast and gray mold, so that the pathogenic bacteria grow and develop abnormally, the sensitivity to the cyanamide is enhanced, and a key technology is provided for reducing the application of pesticides and developing nucleic acid pesticides for preventing and treating plant diseases.

Therefore, the invention provides the application of the myosin5 gene Myo5 segment in preventing and controlling plant fungal diseases and/or enhancing the disease resistance of plants.

And the use of the myosin5 gene Myo5 segment for inhibiting the development and virulence of pathogenic fungi.

And the use of the Myo-5 gene Myo5 segment for increasing the drug sensitivity of pathogenic fungi to myosin inhibitors.

In the present invention, the myosin5 gene is derived from Fusarium graminearum (Fusarium graminearum), Fusarium asiacum (f.asiticum), Fusarium graminearum (f.moniliforme), Fusarium oxysporum (f.oxysporum), pyricularia oryzae (Magnaporthe oryzae), Botrytis cinerea (Botrytis cinerea), Verticillium dahliae (Verticillium dahliae), or Sclerotinia sclerotiorum (sclerotiorum).

In the present invention, the Myo5 segment of the myosin5 gene is a siRNA of 15-30nt generated randomly by RNase digestion with a Myo5dsRNA segment, a combination of dsRNA segments or with full length or partial Myo5 dsRNA.

Wherein the Myo5dsRNA segment is obtained by dividing cDNA of Myo5 gene into 8 different segments, and each segment is named Myo5-1(cDNA start and stop site 1nt-473nt), Myo5-2(cDNA start and stop site 456nt-938nt), Myo5-3(cDNA start and stop site 939nt-1455nt), Myo5-4(cDNA start and stop site 1381nt-1915nt), Myo5-5(cDNA start and stop site 1828nt-2313nt), Myo5-6(cDNA start and stop site 2253nt-2739nt), Myo5-7(cDNA start and stop site 2649nt-3206nt) and Myo5-8 (corresponding cDNA start and stop site 3149-3645 when the segment is derived from Fusarium graminearum (Fusarium graminearum) or Fusarium oryzae (Fusarium oryzae), and Fusarium oryzae (Magnaporthe grisea) or Fusarium oryzae 3149 nt-final base of cDNA start-stop site corresponding to Botrytis cinerea, Verticillium dahliae or Sclerotinia sclerotiorum.

According to a preferred embodiment, the dsRNA segment is a 1200bp Myo5 gene dsRNA segment synthesized by using the full length Myo5cDNA as a template, and the sequence of the dsRNA segment is identical to the sequence of the dsRNA segment after replacing T (thymine) by U (uracil) as a corresponding template.

Taking the Myo5 gene derived from fusarium as an example, the dsRNA segment is constructed by the following steps:

(1) The cDNA of Myo5 gene is divided into 8 different segments, which are respectively named as Myo5-1(cDNA start and stop site: 1nt-473nt), Myo5-2(cDNA start and stop site: 456nt-938nt), Myo5-3(cDNA start and stop site: 939nt-1455nt), Myo5-4(cDNA start and stop site: 1381nt-1915nt), Myo5-5(cDNA start and stop site: 1828nt-2313nt), Myo5-6(cDNA start and stop site: 2253nt-2739nt), Myo5-7(cDNA start and stop site: 2649nt-3206nt) and Myo5-8(cDNA start and stop site: 3149nt-3645 nt), and the forward sequence of each segment amplified by specific PCR primers is designed, wherein the sequences of the primers are as follows:

MRNAi-1F:ggggacaagtttgtacaaaaaagcaggctatgggaatatcgagacgcccgaagaac

MRNAi-1R:ggggaccactttgtacaagaaagctgggttctcccgattctccaccagaca

MRNAi-2F:ggggacaagtttgtacaaaaaagcaggcttggtggagaatcgggagatatca

MRNAi-2R:ggggaccactttgtacaagaaagctgggtaccctggtcttcttggaactga

MRNAi-3F:ggggacaagtttgtacaaaaaagcaggctccagggtggttatgctgaagtc

MRNAi-3R:ggggaccactttgtacaagaaagctgggtgtggcgtccttcatggcggaga

MRNAi-4F:ggggacaagtttgtacaaaaaagcaggctgataacaaggttgtttgcgatc

MRNAi-4R:ggggaccactttgtacaagaaagctgggtaaccggctcttctgatgcgaacg

MRNAi-5F:ggggacaagtttgtacaaaaaagcaggctcgccaacagaatacaacggccc

MRNAi-5R:ggggaccactttgtacaagaaagctgggtgacgcctttccttgcgacctcc

MRNAi-6F:ggggacaagtttgtacaaaaaagcaggctgaatttctccagcttcgtgacc

MRNAi-6R:ggggaccactttgtacaagaaagctgggttgaagccgcctggcatcactcg

MRNAi-7F:ggggacaagtttgtacaaaaaagcaggctggatcaccgcaagaggctgacc

MRNAi-7R:ggggaccactttgtacaagaaagctgggttgggcttggtggccgtgctggg

MRNAi-8F:ggggacaagtttgtacaaaaaagcaggctctggacgagcaccgcctccgcc

MRNAi-8R:ggggaccactttgtacaagaaagctgggtaccagtcatcgtcgtcttccttc

total PCR reaction system 50 μ l: myosin 5cDNA template 1. mu.l, 10 × LA PCRbuffer 5. mu.l, 10mM dNTP 4. mu.l, forward and reverse primers 1. mu.l each, LATaq enzyme 0.5. mu.l, water to 50. mu.l; the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; 35 cycles of 95 ℃ for 20s, 56 ℃ for 30s, 72 ℃ for 30 s; extending for 10min at 72 ℃;

And respectively mixing the PCR product after gel recovery with a commercial pDONR201 donor vector to form an entry vector containing attL1/attL2 new sites, respectively mixing the entry vector with a target vector pDestination to perform specific recombination, and transforming the reaction product into escherichia coli competence to form an interference expression vector of a pMyo5RNAi series: pMyo5RNAi-1, pMyo5RNAi-2, pMyo5RNAi-3, pMyo5RNAi-4, pMyo5RNAi-5, pMyo5RNAi-6, pMyo5RNAi-7 and pMyo5RNAi-8, wherein the interfering expression vectors respectively comprise sections Myo5-1, Myo5-2, Myo5-3, Myo5-4, Myo5-5, Myo5-6, Myo5-7 and Myo5-8, and the sequence of the dsRNA section is the same as that of the section U (uracil) instead of T (thymine).

In a preferred embodiment, the combination of dsRNA segments is a gene segment obtained by using an RNAi Kit, using a combination of a plurality of dsRNA segments as templates for synthesizing the segments.

Furthermore, the invention also provides an in vitro interference preparation, which contains the myoglobulin 5 gene Myo5 segment.

As an alternative embodiment, the preparation method of the formulation is as follows:

(1) formulation of Myo5dsRNA

1) Using the full length of Myo5cDNA as a template to synthesize 4 Myo5 gene dsRNA Myo5-9(1nt-1200nt), Myo5-10(901nt-2100nt), Myo5-11(1801nt-3000nt) and Myo5-12(2446nt-3645nt) with the size of 1200bp, wherein the dsRNA is synthesized by using an RNAi Kit AM1626 Kit of Invitrogen company, and the sequences of the 4 Myo5dsRNA are the same as the sequences of a corresponding template U (uracil) substituted for T (thymine);

2) Using Myo5-1, Myo5-2, Myo5-3, Myo5-4, Myo5-5, Myo5-6, Myo5-7 and Myo5-8 as templates of synthetic segments of in vitro interference dsRNA, wherein the synthetic segments are independently or mutually combined to synthesize 14 Myo5dsRNA, the sequence of the 14 Myo5dsRNA is the same as that of the corresponding template U (uracil) instead of T (thymine) by using RNAi AM1626 Kit of Invitrogen company;

3) Using the Myo5cDNA full length as a template, a full length Myo5dsRNA was synthesized using an RNAi Kit AM1626 Kit from Invitrogen, and digested with RNase III to randomly generate siRNA of 15-30 nt.

The invention also provides application of the in vitro interference preparation in breeding of disease-resistant varieties of transgenic plants.

Aiming at various different types of fungal diseases, such as wheat (barley) scab and powdery mildew, wheat (barley) rust, wheat (corn and rice) sheath blight, verticillium dahliae, sclerotinia sclerotiorum and the like, the invention takes the Myo5 of the fungi or homologous genes thereof as target genes and is applied to the improvement of disease resistance of crops such as wheat, barley, corn and rice and the like and the reduction of pathogenicity and drug resistance of fusarium graminearum, powdery mildew pathogenic bacteria, rust pathogenic bacteria, rice blast pathogenic bacteria and sheath blight pathogenic bacteria, verticillium wilt pathogenic bacteria of cotton and sclerotinia sclerotiorum pathogenic bacteria. Experiments prove that the myosin-5 Myo5 gene RNA interference technology has the outstanding advantages of increasing the drug sensitivity of pathogenic fungi, reducing the drug resistance level, interfering the pathogenicity, enhancing the disease resistance of plants, preventing and treating the specificity of plant diseases and the like, and is green and safe.

[ description of the drawings ]

FIG. 1 is a schematic diagram of the division of Myo5 gene into 8 different segments and the corresponding construction of fungal pMyo5RNAi vector.

FIG. 2 shows the phenotype of the cyanamide resistance strain JT04-1 and different Myo5RNAi strains cultured in PDA medium for 3 d.

FIG. 3 is a Southern bolt test of Myo5RNAi strain.

In the figure: JT04-1 is a negative control strain, and two bands are single copy gene integrative transformants.

FIG. 4 shows the pathogenicity analysis of Myo5RNAi strain inoculated with Yumai 33 seedling stage.

The control is strain JT 04-1.

FIG. 5 is a determination of the prevention effect of a highly antibacterial strain JT04-1 and MyoRNAi transformants by spraying a 25% phenamacril suspending agent in the field.

in the figure: different letters represent significant differences between samples (P < 0.05).

FIG. 6 shows the determination of the effect of Myo5dsRNA on fusarium. Myo5dsRNA and siRNA concentrations shown in the figure are 40 ng/. mu.l.

FIG. 7 shows the analysis of RNA concentration and bacteriostatic efficiency.

FIG. 8 shows the homology analysis of the Myosin5 gene in different fungi.

FIG. 9 is a schematic diagram of an Arabidopsis thaliana transformation vector.

[ detailed description ] embodiments

The following examples serve to illustrate the technical solution of the present invention without limiting it.

in the present invention, "%" and "parts" used for concentration are weight percentages and parts are parts by weight, unless otherwise specified.

The invention relates to the following culture media, which respectively comprise the following components:

PDA culture medium: boiling potato 200g for 15min, collecting the leachate, glucose 20g, agar 15g, and distilled water to 1000ml, and sterilizing at 121 deg.C for 20 min;

SNA culture medium: 0.1% KH2PO4, 0.1% KNO3, 0.05% MgSO4 & lt 7 & gt H2O, 0.05% KCl, 0.02% glucose and 0.02% sucrose, distilled water to a volume of 1000 ml.